Reconstitutable acid solubilized collagen



United States Patent 3,178,301 RECONSTITUTABLE ACID SOLUBILIZED COLLAGENArthur Veis, Skokie, and Jerome Cohen, Chicago, IlL,

assignors, by mesne assignments, to Armour and Company, Chicago, Ill., acorporation of Delaware No Drawing. Filed Sept. 19, 1960, Ser. No.56,646 4 Claims. (Cl. 106-124) This invention relates to collagen fibersand particularly it relates to warm acid solubilized fragments ofcollagen fibers which are susceptible to ready repolymerization andreprecipitation as networks of highly organized long collagen fibers, toa method for preparing them, and to a method for reconstructingcollagenous fibers.

Leather, which consists of chemically modified collagen, is generallyavailable only in form of sheets of intact animal skin. Manufacturers ofleather goods cut pieces of desired shape, size and quality from thesheets of skin and discard the remainder as scrap which is disposed ofin relatively uneconomical ways. Dilterences in quality, thickness andshape of natural leather sheets further add to the difiiculties andamount of scrap encountered by fabricators. It has long been recognizedthat it would be desirable to have methods available for dissolvingcollagen, the solubilized collagen being susceptible to reconstitutionas uniform sheets of leather.

The treatment of collagen to dissolve it generally results in theproduction of material such as gelatin. Generally, gelatin isirreversibly denatured, there being no known practical method forrepolymerizing or reconstituting gelatin or similar materials into thenative-like fibrous structure found in leather.

A scientific fact which has been known for some time is that normallyoccurring collagen contains very minor amounts of a substance calledtropocollagen or procollagen which may be solubilized and reconstitutedinto the native-like fibrous structures found in leather. However, thereconstitutable fraction of native collagen can be obtained only by acareful low temperature extraction procedure; relatively scarce andexpensive collagen starting materials such as calf skins must be used;and use of mature bovine hide often results in extraction of only tracesof tropocollagen.

Attempts to increase the yields of the tropocollagen or procollagen byraising the temperature of the extraction procedure have resulted onlyin total destruction of the material. During heated acid or alkaliextraction of the collagen, the collagen is normally irreversiblydepolymerized to form water soluble gelatin from which no native-likecollagen fibers can be extracted.

There is need for a method of increasing the yield of thetropocollagen-like or procollagen-like materials obtained from collagenwithout destroying the reconstitutable character of the substances.

By the use of one aspect of our invention, we are able to extract frommature steer hide up to about by weight of soluble collagen which isamenable to reaggregation as cross-linked, highly oriented collagenfibers and which has the properties of tropocollagen.

An important object of this invention is to provide a highly elficientand simple method for solubilizing native collagen which is susceptibleto reprecipitation as polymerized fibers of tropocollagen. Anotherobject is to provide collagen material that is acid extractable attemperatures above room temperature and which subsequently displays theproperties of tropocollagen. A further object is to provide a method forprocessing and altering collagen so as to form reconstructed collagenousstructures of desired form and quality. Other objects and advantageswill appear in the following description.

In accordance with this invention solubilized collagen having theproperties of tropocollagen is extracted from native collagen in thepresence of hydroxy acids at temperatures of from about room temperatureto about 70 C.

Collagenous materials from various sources are suitable startingmaterials to produce the products of this invention. Examples of suchmaterials include hides, skins, tendons, ossein and so on. Preferablymature steer hide or hide scraps are used as raw material. We may usecollagen which has been subjected to various pretreatments such asdehydration, salting and the like. Optimally, the collagenous stock isdehaired, fleshed and thoroughly cleaned and washed prior to processing.

In the practice of this invention the collagen is mechanicallysubdivided by a variety of well-known methods, such as beating, grindingor comminuting. One satisfactory method of accomplishing this procedureis to introduce the collagen into a paper beating machine withsufliicient water and with suflicient mixing and beating to form atree-flowing slurry. A preferred type of apparatus is the Hollander typepaper beater which yields a satisfactory slurry without appreciablyreducing the length of the native fibers.

Following mechanical sub-division the disaggregated collagen slurry maythen be solubilized with warm acid. Preferably the fibers are firsttreated to remove part of the water which may have been added during thebeating operation. Wringing the collagen fibers by rubber-rollers is onesuitable method. Hydroxylated organic acids produce the dissolvedreprecipitatable collagen which is important in this invention. Alkyl,aryl and heterocyclic hydroxylated carboxy and polycarboxy acids whichmay be dissolved in water to give solutions having pI-Is between about2.5 and 4.0 are suitable reagents for this solubilization. Although weprefer to employ citric acid in the process, examples of other acidswhich may be used include benzilic, glycolic, glycerolphosphoric,hydroxybutyric, lactic, and the like. Acids of this type are believed toreact with the collagen fibers during heating and solubilization therebypreventing protein reaction with water present, as a consequence ofwhich the collagen fiber fragments cannot collapse. The dissolved fibersthus remain susceptible to reprecipitation in a native cross-linkedpolymeric pattern.

Concentration of the acid should be suflicient to give a pH of about 2.5to 4.0 in the collagen-acid solution reaction mixture. Expresseddifferently, this pH will generally be obtained by employing about 0.01molar to 1 molar acid solution, depending on the individual acid or acidmixture utilized. We prefer to use acid concentrations giving a reactionmixture pH of about 3.0 to 3.5. When reacting slurries which have beende-watered to yield wet fibers composed of about 25% protein and water,one part of collagen fibers to about one and onehalf parts by weight of0.1 molar citric acid gives a reaction mixture with satisfactoryacidity.

Throughout the solubilizing portion of our process, it

is most important that temperature of the reaction mix-V ture bemaintained between about room temperature and 70 C. Best results areusually experienced when a tem perature of between about 30 C. and 60 C.is used. Poor yields of soluble reprecipitatable col-lagen fiberfragments normally result from the use of temperatures much below roomtemperatures. Temperatures above 70 C. should be avoided as gelatin maybe formed in large quantities and we prefer not to form it in ourprocess.

When the heated and acidified fiber mass has been properly prepared itwill assume a physical state which may be described as stringyplasticity. Holding the warm acidified collagen for about 30 to 60minutes will usually be adequate for proper solubilization. Loweracidities and Patented Apr. 13, 1965 Q as temperatures may necessitatelonger times, as for example, a temperature of 30 C. at about pH 3.0 mayrequire a reaction time of as much as 3 to 4 hours. The fiber dough ispreferably agitated and blended during solubilization, a screw typeblender giving good results.

The soluble portion of thefiber dough may be extracted andreprecipitated as skin-like or sheet-like structures without thepresence of intact fibers, but we prefer to mix the treated fiber doughcontaining the dissolved collagen with wet fibers which have simply beenmechanically subdivided. Preferably, the wet fibers are lightly tannedby contact with a solution of formaldehyde having a concentration ofabout 0.3 to 3 percent by weight. About percent fiber dough mixed withabout 85 percent by weight of lightly tanned wet fibers and with anequal amount of 'water added provides an excellent mix for preparingreaggregated structures such as sheets, threads and various other forms.The wet mix may then be formed as desired and allowed to remain in acool place for a short time. Thirty minutes at 4 C. is preferred by usbut there is nothing critical about this procedure. The forms, such assheets, may then be reaggregated into stable cross-linked structures byany of several known methods which reprecipitate the solubletropocollagen like collagen fraction. These include heat coagulation,neutralization and salting out of the soluble matter with any of severalreagents. We find good results are obtained by a combination ofneutralization and salting. This is easily accomplished by placing thefiber structure in half saturated ammonium sulphate made basic withammonium hydroxide. The resulting wet skin-like structure maybe furtherprocessed as typical native collagen.

Reprecipitated collagen materials containing the tropocollagen-likefibers of our invention may be formed into varying articles such assheets, threads and other fabricated structure.

The tropocollagendike fibers which are produced as described above havethe following properties. The fibers when viewed by means of an electronmicroscope exhibit the typical cross striations of native collagen,having an axial periodicity of about 600 to 650 A. The materialscontaining the tropocollagen-like fibers of our invention consist ofinterwoven networks of fibers, exhibit reversible synersis, dissolve inwater at pI-Is of about 1 to 3 and are substantially insoluble in waterat pHs of about 4 to 8. In addition these materials have relatively highwet tensile strength.

Our invention provides a method for extracting reprecipitatable solublecollagen from mature mammalian collagen in far greater yields than anymethod heretofore developed. This invention allows the use of cheaperand more readily available raw materials. Moreover, the criticality ofknown methods of extraction is reduced by the use of our process.

The following detailed examples are given only for the purpose ofillustrating and aiding in understanding the invention, it beingunderstood that our invention is not limited to the particularconditions and materials set forth therein.

Example I 1200 gms. of fresh steer hide corium were beaten in a paperbeater with added water. The beaten collagen slurry was dewatered bywringing out the fibers between rubber rollers to give wet fiberscontaining protein and 75% water. 1200 gms. of the wet fibers were addedto 1800 cc. of 0.1 M citric acid solutions. The reaction mixture whichhad a pH of 3.5, was maintained at a temperature of 50 C. for minuteswhile being continuously mixed in a screw type blender. 450 gms. of theacid treated fiber dough were added to 940 gms. of wet fibers, which hadbeen de-watered, and the mixture was lightly tanned in 3% formaldehydesolution for a short period of time. To the lightly tanned wet fiberswas added 450 gms. of fiber dough and 1505 cc. of water. The fiber andwater mixture were blended for 30 minutes and rolled into sheets havinga thickness of about .016 inch. The sheet was placed in a cold room atabout 4 C. for 30 minutes after which it was immersed in half saturatedammonium sulphate which had been adjusted to pH 8.1 by the addition ofammonium hydroxide. The ammonium sulphate solution was prepared byadding 1100 cos. of water to one pound of ammonium sulphate. After fourhours immersion the sheets were water-washed and chrome tanned toproduce a high strength leather-like product.

Example 11 Bovine corium collagen pieces of about 1 cm. cubed weresuspended in 0.1 M citric acid at room temperature. After the collagenpieces were wet through with citric acid solution, the mixture washeated with stirring to 50 C. for one hour. The majority of the materialdissolved, leaving a few pieces of individual fibers remaining. Theentire slurry was blended in a Waring blender to obtain uniformdispersion. This slurry was then cooled to room temperature and extrudedthrough an orifice onto a moving belt which carried slurry through aone-half saturated solution of ammonium sulfate. pH of bath wasmaintained at 8.5 with ammonium hydroxide. Extruded fibers wer carriedthrough several feet of the bath and then picked up on a second conveyorbelt and transferred to a bath of cold water maintained at low ionicstrength. As soon as salt was removed, fibers were picked out of thewater wash and transferred to a drying tower.

Example III Beef corium which had been cleaned and cubed was suspendedin 0.1 M glycolic acid at room temperature. The acidified collagen washeated to 55 C. with constant agitation and held for one hour at thattemperature. The collagenous mix was centrifuged and the supernatantcollected. This supernatant was poured into a pan and chilled at 4 C.for thirty minutes, after which a onehalf saturated solution of ammoniumsulfate adjusted to pH 8.1 with ammonium hydroxide was layered onto theresulting chilled collagen gel. After one hour at room temperature theresulting sheet was dialyzed against tap Water to remove ammoniumsulfate. A high wet strength sheet suitable for tanning resulted.

Example IV Steer hide corium was suspended in 0.15 M citric acid andheated to 50 C. for one hour. The solubilized collagen was put in adialysis bag and dialyzed overnight against cold tap water. A rigid gelwhich formed in the dialysis bag air dried to form a fibrous tubularstructure having high wet strength and which upon examination displayedthe fine structure of native collagen.

Example V Steer hide corium was extracted by the procedure of Example111 with 0.025 M of citric acid at 50 C. for one hour. Approximately a1% yield of solubilized reconstitutable collagen resulted from thedilute hot acid extraction.

Example VI Steer hide was extracted with 0.1 M citric acid solution at60 C. for thirty minutes. A 10% yield of reconstitutable collagen wasrecovered from the collagen thus solubilized. The reconstituted fibersexhibited typical 640 A. banding of native collagen fibers.

Having described our invention what we claim is:

1. The method of preparing reconstitutable solubilized collagen,comprising extracting a slurry of separated native collagen fibers in anaqueous solution of an organic acid at a temperature about 30 C., butnot over 70 C. to solubilize the collagen in said fibers, said organicacid having a concentration within the range from .01 to 1 molar andbeing selected from the group consisting of citric acid, glycolic acid,lactic acid, hydroxybutyric acid, benzilic acid, and glycerol phosphoricacid, said extraction being carried out and completed in a period ofless than four hours, thereby obtaining an enhanced proportion ofsolubilized collagen in reconstitutable form, and subsequentlyreconstituting said solubilized collagen to fibrous collagen having thecross-striations of native collagen.

2. The method of preparing reconstitutable solubilized collagen,comprising extracting a slurry of separated native collagen fibers in anaqueous solution of an organic acid at a temperature of around 50 to 55C. to solubilize the collagen in said fibers, said aqueous solutionhaving a pH of from 2.5 to 4.0 and said organic acid being selected fromthe group consisting of citric acid, glycolic acid, lactic acid,hydroxybutyric acid, benzilic acid, and glycerol phosphoric acid, saidextraction being carried out and completed in a period of not over onehour, thereby obtaining an enhanced proportion of solubilized collagenin reconstitutable form, and subsequently reconstituting saidsolubilized collagen to fibrous collagen having the crossstriations ofnative collagen.

3. The method of preparing reconstitutable solubilized collagen,comprising extracting a slurry of separated native collagen fibers in anaqueous solution of citric acid having a pH of from 2.5 to 4.0 and beingat a temperature above 30 C. but not over 70 C. to solubilize thecollagen in said fibers, said extraction being carried out and completedin a period of less than four hours, thereby obtaining an enhancedproportion of solubilized collagen in reconstitutable form, andsubsequently reconstituting said solubilized collagen to fibrouscollagen having the cross-striations of native collagen.

4. The method of preparing reconstitutable solubilized collagencomprising, extracting a slurry of separated References Cited by theExaminer UNITED STATES PATENTS 2,521,704 9/50 Evans et al 18-542,897,044 7/59 Wormell 18-54 2,934,446 4/60 Highberger et al 106-1552,934,447 4/ Highberger et al 106155 3,071,483 1/63 Tung Tu 1061553,073,702 1/63 Keil et al. 106155 FOREIGN PATENTS 606,427 8/48 GreatBritain.

OTHER REFERENCES Progress in Leather Science 1920-1945, British LeatherManufacturers Research Association, London, England, 1948, pp. 78-80.

Gustavson: The Chemistry and Reactivity of Collagen, Academic PressInc., New York, 1956, pp. 73-77.

OFlaherty et al.: Technology of Leather, vol. 1, pp. 151-162 (1956) (p.155 relied upon).

MORRIS LIEBMAN, Primary Examiner.

CHARLES B. PARKER, MORRIS O. WOLK, LESLIE H. GASTON, Examiners.

1. THE METHOD OF PREPARING RECONSTITUTABLE SOLUBILIZED COLLAGEN,COMPRISING EXTRACTING A SLURRY OF SEPARATED NATIVE COLLAGEN FIBERS IN ANAQUEOUS SOLUTION OF AN ORGANIC ACID AT A TEMPERATURE ABOUT 30*C., BUTNOT OVER 70*C. TO SOLUBILIZE THE COLLAGEN IN SAID FIBERS, SAID ORGANICACID HAVING A CONCENTRATION WITHIN THE RANGE FROM .01 TO 1 MOLAR ANDBEING SELECTED FROM THE GROUP CONSISTING OF CITRIC ACID, GLYCOLIC ACID,LACTIC ACID, HYDROXYBUTYRIC ACID, BENZILIC ACID, AND GLYCEROL PHOSPHORICACID, SAID EXTRATION BEING CARRIED OUT AND COMPLETED IN A PERIOD OF LESSTHAN FOUR HOURS, THEREBY OBTAINING AN ENHANCED PROPORTION OF SOLUBILIZEDCOLLAGEN IN RECONSTITUTABLE FORM, AND SUBSEQUENTLY RECONSTITUTING SAIDSOLUBILIZED COLLAGEN TO FIBROUS COLLAGEN HAVING THE CROSS-STRIATIONS OFNATIVE COLLAGEN.